Fitc conjugated hla dr

Immunophnotypes were evaluated using mouse anti-human monoclonal antibodies: fluorescein isothiocyanate (FITC)-conjugated HLA-DR (BD Biosciences, San Jose, CA) and Phycoerythin (PE)-conjugated CD73, CD90, CD105, CD11b, CD34, CD45 and CD79a. As a control, isotype PE-conjugated IgG1 and FITC-conjugated IgG2a (BD Biosciences) were used. The cell suspension containing 1 × 10⁶ cells was incubated with monoclonal antibodies for 15 minutes at room temperature in the dark and fixed with BD Cytofix^TM (BD Biosciences). The analysis was performed on FACSCalibur cytometer (BD Biosciences) and the resulting data were processed using CellQuest^TM Proversion 6.0 software (BD Biosciences).

Cells harvested from 3rd passage (P3) were used for immunophenotyping analysis. Mouse anti-human monoclonal antibodies: phycoerythrin (PE)-conjugated CD73, CD90, CD105, CD45, CD11b, CD34, CD79a, and FITC-conjugated HLA-DR (BD Biosciences, San Jose, CA) were used. As a control, isotype PE-conjugated IgG1 and FITC-conjugated IgG2a (BD Biosciences) were used. The cell suspension, containing 1 × 10⁶ cells, were incubated with monoclonal antibodies at room temperature in the dark and fixed with BD Cytofix^TM (BD Biosciences). The samples were analyzed on FACSCalibur cytometer (BD Biosciences) and the resulting data were processed using CellQuest^TM Proversion 6.0 software (BD Biosciences).

Phorbol 12-myristate 13-acetate (PMA), Lipopolysaccharides (LPS) and Hoechst 33258 dye were from Sigma-Aldrich (St. Louis, MO, USA). The cytokines for interferon γ (IFNγ), interleukin-4 (IL-4) and interleukin-13 (IL-13) were from R&D Systems (Minneapolis, MN, USA). The antibody for CD163 was from Abcam (Cambridge, MA, USA). The primary antibodies for E-cadherin, GAPDH were from Cell Signaling Technology (Danvers, MA, USA). Antibodies for fluorescein isothiocyanate (FITC)-conjugated CD68, FITC-conjugated CD206, FITC-conjugated CD163, FITC-conjugated HLA-DR and isotype control were from BD Biosciences (San Jose, CA, USA). FITC-conjugated E-cadherin antibody was from BioLegend (San Diego, CA, USA). Hepatocyte maintenance medium (HMM) and SingleQuots were from Lonza (Anaheim, CA, USA).